Novel anti-inflammatory androstane derivatives

ABSTRACT

According to one aspect of the invention, there is provided a pharmaceutical formulation for administration by inhalation comprising a compound of formula (I),  
                 
 
     wherein  
     R 1  represents C 1-6  alkyl or C 1-6  haloalkyl;  
     R 2  represents —C(═O)-aryl or —C(═O)-heteroaryl;  
     R 3  represents hydrogen, methyl (which may be in either the α or β configuration) or methylene;  
     R 4  and R 5  are the same or different and each represents hydrogen or halogen; and  
       represents a single or a double bond;  
     and salts and solvates thereof together with a long-acting β 2 -adrenoreceptor agonist which formulation has a therapeutically useful effect in the treatment of COPD over a period of 24 hours or more.

[0001] This is a divisional application of U.S. Ser. No. 10/066,961,filed Feb. 4, 2002, which is a Continuation-in-part of InternationalPatent Application Serial No. PCT.GB01.03499 filed Aug. 3, 2001, basedupon United Kingdom Patent Application No. GB0108800.4 filed Apr. 7,2001.

[0002] The present invention relates to novel anti-inflammatory andanti-allergic compounds of the androstane series and to processes fortheir preparation. The present invention also relates to pharmaceuticalformulations containing the compounds and to therapeutic uses thereof,particularly for the treatment of inflammatory and allergic conditions.

[0003] Glucocorticoids which have anti-inflammatory properties are knownand are widely used for the treatment of inflammatory disorders ordiseases such as asthma and rhinitis. For example, U.S. Pat. No.4,335,121 discloses 6α,9α-Difluoro-17α-(1-oxopropoxy)-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester (known by the generic name of fluticasonepropionate) and derivatives thereof. The use of glucocorticoidsgenerally, and especially in children, has been limited in some quartersby concerns over potential side effects. The side effects that arefeared with glucocorticoids include suppression of theHypothalamic-Pituitary-Adrenal (HPA) axis, effects on bone growth inchildren and on bone density in the elderly, ocular complications(cataract formation and glaucoma) and skin atrophy. Certainglucocorticoid compounds also have complex paths of metabolism whereinthe production of active metabolites may make the pharmacodynamics andpharmacokinetics of such compounds difficult to understand. Whilst themodern steroids are very much safer than those originally introduced itremains an object of research to produce new molecules which haveexcellent anti-inflammatory properties, with predictable pharmacokineticand pharmacodynamic properties, with an attractive side effect profile,and with a convenient treatment regime.

[0004] We have identified a novel series of glucocorticoids, whichsubstantially meets these objectives.

[0005] Thus, according to one aspect of the invention, there is provideda compound of formula (I)

[0006] wherein

[0007] R₁ represents C₁₋₆ alkyl or C₁₋₆ haloalkyl;

[0008] R₂ represents —C(═O)-aryl or —C(═O)-heteroaryl;

[0009] R₃ represents hydrogen, methyl (which may be in either the α or βconfiguration) or methylene;

[0010] R₄ and R₅ are the same or different and each represents hydrogenor halogen; and

[0011]

represents a single or a double bond;

[0012] and salts and solvates thereof.

[0013] References to the term “aryl” include references to phenyl whichmay be optionally substituted with one or more substituents.

[0014] References to the term “heteroaryl” include references to 5 or 6membered heterocyclic aromatic rings containing 1-3 hetero atomsselected from N, O and S (e.g. pyridinyl, pyrimidinyl, thienyl (egthien-2-yl or thien-3-yl), furanyl (eg furan-2-yl or furan-3-y),pyrrolyl (eg 1H-pyrrol-2-yl), oxazolyl, thiadiazolyl (eg1,2,3-thiadiazol-5-yl, 1,2,5-thiadiazol-3-yl or 1,2,3-thiadiazol-4-yl)).In one respect the heterocycle is preferably thiophenyl, pyrrolyl orfuranyl, more preferably thiophenyl or furanyl. Further examples includethiazolyl (eg 1,3-thiazolyl-5-yl or 1,3-thiazolyl-4-yl), isoxazolyl (egisoxazol-5-yl or isoxazol-4-yl), isothiazolyl (eg isothiazol-3-yl orisothiazol-5-yl), pyrazolyl (eg 1H-pyrazol-5-yl) and imidazolyl (eg1H-imidazol-5-yl). All of the previously named heterocycles may beoptionally substituted with one or more (e.g. 1 or 2) substituents.

[0015] Examples of substituents for aryl and heteroaryl include C₁₋₆alkyl (e.g. methyl) or halogen (e.g. chlorine or bromine). Otherexamples include C₁₋₆ alkoxy (e.g. methoxy or ethoxy).

[0016] Examples of substituted furanyl include 3-Me-furan-2-yl,5-Br-furan-2-yl, 2-Me-furan-3-yl and 2,5-diMe-furan-3-yl. Examples ofsubstituted thienyl include 3-Me-thien-2-yl, 5-Me-thien-2-yl,5-Cl-thien-2-yl, 3—Cl-thien-2-yl, 3-Br-thien-2-yl, 3-Ethoxy-thien-2-yl,4-methoxy-thien-3-yl, 2,5-diCl-thien-3-yl and 4-methoxy-5—Cl-thien-3-yl.Examples of substituted pyrrolyl include 1-Me-1H-pyrrol-2-yl. Examplesof substituted thiazolyl include 4-Me-1,3-thiazol-5-yl,2,4-diMe-1,3-thiazol-5-yl and 2-Me-1,3-thiazol-4-yl. Examples ofsubstituted thiadiazolyl include 4-Me-1,2,3-thiadiazol-5-yl. Examples ofsubstituted isoxazolyl include 3-Me-isoxazol-5-yl, 5-Me-isoxazol-3-yl,5-Me-isoxazol -4-yl and 3,5-diMe-isoxazol-4-yl. Examples of substitutedpyrazolyl include 1,3-diMe -1H-pyrazol-5-yl andi-Et-3-Me-1H-pyrazol-5-yl. Examples of substituted imidazolyl include1-Me-1H-imidazol-5-yl.

[0017] Examples of solvates include hydrates.

[0018] Examples of salts of compounds of formula (I) includephysiologically acceptable salts which may be formed with basiccompounds (such as when heteroaryl is basic) eg. acetate, benzoate,citrate, succinate, lactate, tartrate, fumarate and maleate.

[0019] References hereinafter to a compound according to the inventionincludes both compounds of formula (I) and salts and solvates thereof,particularly pharmaceutically acceptable salts and solvates.

[0020] It will be appreciated that the invention includes within itsscope all stereoisomers of the compounds of formula (I) and mixturesthereof.

[0021] Preferably, the absolute stereochemistry will be as shown in therepresentation of compounds of formula (I).

[0022] Examples of C₁₋₆ haloalkyl that R₁ may represent include C₁₋₆alkyl substituted by 1-3 halogen atoms, preferably 1 halogen atom.Preferred halogen atoms are selected from bromine, chlorine andfluorine. Examples of C₁₋₆ alkyl that R₁ may represent include methyl.

[0023] We prefer R₁ to represent fluoromethyl, chloromethyl, bromomethylor 2′-fluoroethyl, especially fluoromethyl.

[0024] We prefer R₂ to represent —C(═O)-heteroaryl. Preferably theheteroaryl is a 5-membered heterocyclic aromatic ring containing 1 to 3heteroatoms selected from O, N and S which may optionally besubstituted. In one respect, preferably heteroaryl represents furanyl,pyrrolyl or thiophenyl, more preferably furanyl or thiophenyl eg2-furanyl, 3-furanyl, 2-thiophenyl or 3-thiophenyl, especially furanyl,particularly 2-furanyl. However it may also be preferred that heteroarylrepresents pyrrolyl or thiophenyl, more preferably thiophenyl eg2-thiophenyl or 3-thiophenyl which pyrrolyl or thiophenyl may beoptionally substituted.

[0025] Of particular interest are compounds in which the heteroaryl is a5-membered heterocyclic aromatic ring containing 2 heteroatoms selectedfrom O, N and S. Thus a further set of preferred compounds is that inwhich R₂ represents optionally substituted thiazolyl. A further set ofpreferred compounds is that in which R₂ represents optionallysubstituted isothiazolyl. A further set of preferred compounds is thatin which R₂ represents optionally substituted pyrazolyl. A further setof preferred compounds is that in which R₂ represents optionallysubstituted isoxazolyl. A further set of preferred compounds is that inwhich R₂ represents optionally substituted isothiazolyl. A further setof preferred compounds is that in which R₂ represents optionallysubstituted imidazolyl.

[0026] Of particular interest is compounds in which the heteraryl is a5-membered heterocyclic aromatic ring containing 3 heteroatoms selectedfrom O, N and S. Thus a further set of preferred compounds is that inwhich R₂ represents optionally substituted thiadiazolyl.

[0027] Compounds in which R₂ represents —C(═O)-aryl (preferably arylrepresents phenyl) are also of particular interest.

[0028] We prefer R₃ to represent methyl, especially methyl in the αconfiguration. Compounds in which R₃ represents methyl in the,configuration are also of particular interest.

[0029] Compounds of formula (I) in which R₄ and R₅, which can be thesame or different, each represents hydrogen, fluorine or chlorine,particularly hydrogen or fluorine, are preferred. Especially preferredare compounds in which both R₄ and R₅ are fluorine.

[0030] Preferably,

[0031] represents a double bond. Compounds in which

[0032] represents a single bond are also of particular interest.

[0033] A particularly preferred group of compounds of the presentinvention are compounds of formula (I) in which R₁ is fluoromethyl; R₂is —C(═O)-2-furanyl; R₃ is methyl; R₄ and R₅, which can be the same ordifferent, each represents hydrogen or fluorine, especially fluorine,and

[0034] represents a single or a double bond.

[0035] It is to be understood that the present invention covers allcombinations of particularly and preferred groups referred tohereinabove.

[0036] Preferred compounds of formula (I) include:

[0037]6α,9α-Difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester;

[0038]6α,9α-Difluoro-17β-[(3-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester;

[0039]6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(2-thienylcarbonyl)oxy]-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester;

[0040]6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(3-thienylcarbonyl)oxy]-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester;

[0041]17α-(Benzoyl)oxy-6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester;

[0042]9α-Fluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16β-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester; and

[0043]6α,9α-Difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androst-4-ene-17β-carbothioic acid S-fluoromethyl ester or a salt or solvatethereof.

[0044] Particularly preferred compounds of formula (I) include:

[0045]6α,9α-Difluoro-17αα-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester;

[0046]6α,9α-Difluoro-17α-[(3-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester;

[0047]6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(2-thienylcarbonyl)oxy]-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester;

[0048]6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(3-thienylcarbonyl)oxy]-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester;

[0049]6α,9α-Difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androst-4-ene-17β-carbothioic acid S-fluoromethyl ester or a salt or solvatethereof.

[0050] The compounds named as Examples 8-43 are also of particularinterest, especially Examples 8-22 and 24-43, most especially examples8-9, 11-13, 15-22 and 24-43.

[0051] The compounds of formula (I) have potentially beneficialanti-inflammatory or anti-allergic effects, particularly upon topicaladministration, demonstrated by, for example, their ability to bind tothe glucocorticoid receptor and to illicit a response via that receptorwith long lasting effect. Hence, the compounds of formula (I) are usefulin the treatment of inflammatory and/or allergic disorders, especiallyin once-per-day therapy.

[0052] Compounds of formula (I) are predicted to undergo highlyefficient hepatic metabolism to yield the corresponding 17-β carboxylicacid (X) in which R₂-R₄ and

[0053] are as defined above as the sole major metabolite in rat andhuman in vitro systems. We have established that this is the case forExample 1 and metabolite (X) for Example 1 has been synthesised anddemonstrated to be>1000 fold less active than the parent compound in invitro functional glucocorticoid agonist assays. Analogues of (X) whereinR₂ represents a group other than 2-furanylcarbonyl are expected also tohave very low activity.

[0054] This efficient hepatic metabolism is reflected by in vivo data inthe rat for certain examples, which have demonstrated plasma clearanceat a rate approaching hepatic blood flow (Examples 1, 4, 19, 24, 25 and28) and an oral bioavailability of <1%, consistent with extensivefirst-pass metabolism (Example 1).

[0055] In vitro metabolism studies in human hepatocytes havedemonstrated that Example 1 is metabolised in an identical manner tofluticasone propionate but that conversion of Example 1 to the inactiveacid metabolite occurs approximately 5-fold more rapidly than withfluticasone propionate. This very efficient hepatic inactivation wouldbe expected to minimise systemic exposure in man leading to an improvedsafety profile.

[0056] Inhaled steroids are also absorbed through the lung and thisroute of absorption makes a significant contribution to systemicexposure. Reduced lung absorption could therefore provide an improvedsafety profile. Studies with Example 1 have shown significantly lowerexposure to Example 1 than with fluticasone propionate after dry powderdelivery to the lungs of anaesthetised pigs.

[0057] An improved safety profile is believed to allow the compounds offormula (I) to demonstrate the desired anti-inflammatory effects whenadministered once-per day. Once-per-day dosing is considered to besignificantly more convenient to patients than the twice-per day dosingregime that is normally employed for fluticasone propionate.

[0058] Examples of disease states in which the compounds of theinvention have utility include skin diseases such as eczema, psoriasis,allergic dermatitis neurodermatitis, pruritis and hypersensitivityreactions; inflammatory conditions of the nose, throat or lungs such asasthma (including allergen-induced asthmatic reactions), rhinitis(including hayfever), nasal polyps, chronic obstructive pulmonarydisease, interstitial lung disease, and fibrosis; inflammatory bowelconditions such as ulcerative colitis and Crohn's disease; andauto-immune diseases such as rheumatoid arthritis.

[0059] Compounds of the invention may also have use in the treatment ofconjunctiva and conjunctivitis.

[0060] It will be appreciated by those skilled in the art that referenceherein to treatment extends to prophylaxis as well as the treatment ofestablished conditions.

[0061] As mentioned above, compounds of formula (I) are useful in humanor veterinary medicine, in particular as anti-inflammatory andanti-allergic agents.

[0062] There is thus provided as a further aspect of the invention acompound of formula (I) or a physiologically acceptable salt or solvatethereof for use in human or veterinary medicine, particularly in thetreatment of patients with inflammatory and/or allergic conditions,especially for treatment once-per-day.

[0063] According to another aspect of the invention, there is providedthe use of a compound of formula (1) or physiologically acceptable saltor solvate thereof for the manufacture of a medicament for the treatmentof patients with inflammatory and/or allergic condition, especially fortreatment once-per-day.

[0064] In a further or alternative aspect, there is provided a methodfor the treatment of a human or animal subject with an inflammatoryand/or allergic condition, which method comprises administering to saidhuman or animal subject an effective amount of a compound of formula (I)or physiologically acceptable salt or solvate thereof, especially foradministration once-per-day.

[0065] The compounds according to the invention may be formulated foradministration in any convenient way, and the invention therefore alsoincludes within its scope pharmaceutical compositions comprising acompound of formula (I) or physiologically acceptable salt or solvatethereof together, if desirable, in admixture with one or morephysiologically acceptable diluents or carriers. Pharmaceuticalcompositions suitable for once-per-day administration are of particularinterest.

[0066] Further, there is provided a process for the preparation of suchpharmaceutical compositions which comprises mixing the ingredients.

[0067] The compounds according to the invention may, for example, beformulated for oral, buccal, sublingual, parenteral, local or rectaladministration, especially local administration.

[0068] Local administration as used herein, includes administration byinsufflation and inhalation. Examples of various types of preparationfor local administration include ointments, lotions, creams, gels,foams, preparations for delivery by transdermal patches, powders,sprays, aerosols, capsules or cartridges for use in an inhaler orinsufflator or drops (e.g. eye or nose drops), solutions/suspensionsformebulisation, suppositories, pessaries, retention enemas and chewableor suckable tablets or pellets (e.g. for the treatment of aphthousulcers) or liposome or microencapsulation preparations.

[0069] Advantageously compositions for topical administration to thelung include dry powder compositions and spray compositions.

[0070] Dry powder compositions for topical delivery to the lung may, forexample, be presented in capsules and cartridges for use in an inhaleror insufflator of, for example, gelatine. Formulations generally containa powder mix for inhalation of the compound of the invention and asuitable powder base such as lactose or starch. Use of lactose ispreferred. Each capsule or cartridge may generally contain between 20μg-10 mg of the compound of formula (I) optionally in combination withanother therapeutically active ingredient. Alternatively, the compoundof the invention may be presented without excipients. Packaging of theformulation may be suitable for unit dose or multi-dose delivery. In thecase of multi-dose delivery, the formulation can be pre-metered (eg. asin Diskus, see GB 2242134 or Diskhaler, see GB 2178965, 2129691 and2169265) or metered in use (eg. as in Turbuhaler, see EP 69715). Anexample of a unit-dose device is Rotahaler (see GB 2064336). The Diskusinhalation device comprises an elongate strip formed from a base sheethaving a plurality of recesses spaced along its length and a lid sheethermetically but peelably sealed thereto to define a plurality ofcontainers, each container having therein an inhalable formulationcontaining a compound of formula (I) optionally in combination withanother therapeutically active ingredient preferably combined withlactose. Preferably, the strip is sufficiently flexible to be wound intoa roll. The lid sheet and base sheet will preferably have leading endportions which are not sealed to one another and at least one of thesaid leading end portions is constructed to be attached to a windingmeans. Also, preferably the hermetic seal between the base and lidsheets extends over their whole width. The lid sheet may preferably bepeeled from the base sheet in a longitudinal direction from a first endof the said base sheet.

[0071] Spray compositions may for example be formulated as aqueoussolutions or suspensions or as aerosols delivered from pressurisedpacks, such as a metered dose inhaler, with the use of a suitableliquefied propellant. Aerosol compositions suitable for inhalation canbe either a suspension or a solution and generally contain the compoundof formula (I) optionally in combination with another therapeuticallyactive ingredient and a suitable propellant such as a fluorocarbon orhydrogen-containing chlorofluorocarbon or mixtures thereof, particularlyhydrofluoroalkanes, especially 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. The aerosolcomposition may optionally contain additional formulation excipientswell known in the art such as surfactants e.g. oleic acid or lecithinand cosolvents e.g. ethanol. One example formulation is excipient freeand consists essentially of (eg consists of) a compound of formula (I)(optionally together with another active ingredient) and a propellantselected from 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane and mixture thereof. Another exampleformulation comprises particulate compound of formula (I), a propellantselected from 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane and mixture thereof and a suspending agent whichis soluble in the propellant eg an oligolactic acid or derivativethereof as described in WO94/21229. The preferred propellant is1,1,1,2-tetrafluoroethane. Pressurised formulations will generally beretained in a canister (eg an aluminium canister) closed with a valve(eg a metering valve) and fitted into an actuator provided with amouthpiece.

[0072] Medicaments for administration by inhalation desirably have acontrolled particle size. The optimum particle size for inhalation intothe bronchial system is usually 1-10μm, preferably 2-5μm. Particleshaving a size above 20μm are generally too large when inhaled to reachthe small airways. To achieve these particle sizes the particles ofcompound of formula (I) (and any further therapeutically activeingredient) as produced may be size reduced by conventional means eg. bymicronisation. The desired fraction may be separated out by airclassification or sieving. Preferably, the particles will becrystalline, prepared for example by a process which comprises mixing ina continuous flow cell in the presence of ultrasonic radiation a flowingsolution of compound of formula (I) as medicament in a liquid solventwith a flowing liquid antisolvent for said medicament (eg as describedin International Patent Application PCT/GB99/04368) or else by a processwhich comprises admitting a stream of solution of the substance in aliquid solvent and a stream of liquid antisolvent for said substancetangentially into a cylindrical mixing chamber having an axial outletport such that said streams are thereby intimately mixed throughformation of a vortex and precipitation of crystalline particles of thesubstance is thereby caused (eg as described in International PatentApplication PCT/GB00/04327). When an excipient such as lactose isemployed, generally, the particle size of the excipient will be muchgreater than the inhaled medicament within the present invention. Whenthe excipient is lactose it will typically be present as milled lactose,wherein not more than 85% of lactose particles will have a MMD of 60-90μm and not less than 15% will have a MMD of less than 15 μm.

[0073] Formulations for administration topically to the nose includepressurised aerosol formulations and aqueous formulations administeredto the nose by pressurised pump.

[0074] Aqueous formulations for administration to the lung or nose maybe provided with conventional excipients such as buffering agents,tonicity modifying agents and the like. Aqueous formulations may also beadministered to the nose by nebulisation.

[0075] Other possible presentations include the following:

[0076] Ointments, creams and gels, may, for example, be formulated withan aqueous or oily base with the addition of suitable thickening and/orgelling agent and/or solvents. Such bases may thus, for example, includewater and/oran oil such as liquid paraffin or a vegetable oil such asarachis oil or castor oil, or a solvent such as polyethylene glycol.Thickening agents and gelling agents which may be used according to thenature of the base include soft paraffin, aluminium stearate,cetostearyl alcohol, polyethylene glycols, woolfat, beeswax,carboxypolymethylene and cellulose derivatives, and/or glycerylmonostearate and/or non-ionic emulsifying agents.

[0077] Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilisingagents, dispersing agents, suspending agents or thickening agents.

[0078] Powders for external application may be formed with the aid ofany suitable powder base, for example, talc, lactose or starch. Dropsmay be formulated with an aqueous or non-aqueous base also comprisingone or more dispersing agents, solubilising agents, suspending agents orpreservatives.

[0079] If appropriate, the formulations of the invention may be bufferedby the addition of suitable buffering agents.

[0080] The proportion of the active compound of formula (I) in the localcompositions according to the invention depends on the precise type offormulation to be prepared but will generally be within the range offrom 0.001 to 10% by weight. Generally, however for most types ofpreparations advantageously the proportion used will be within the rangeof from 0.005 to 1% and preferably 0.01 to 0.5%. However, in powders forinhalation or insufflation the proportion used will usually be withinthe range of from 0.1 to 5%.

[0081] Aerosol formulations are preferably arranged so that each metereddose or “puff” of aerosol contains 1 μg-2000 μg eg 20 μg-2000 μg,preferably about 20 μg-500 μg of a compound of formula (I) optionally incombination with another therapeutically active ingredient.Administration may be once daily or several times daily, for example 2,3, 4 or 8 times, giving for example 1, 2 or 3 doses each time.Preferably the compound of formula (I) is delivered once or twice daily,especially once per day. The overall daily dose with an aerosol willtypically be within the range 10 μg-10 mg eg 100 μg-10 mg preferably,200 μg-2000 μg.

[0082] Since the compounds of formula (I) are long-acting, preferablythe compound will be delivered once-per-day and the dose will beselected so that the compound has a therapeutic effect in the treatmentof respiratory disorders (eg asthma or COPD, particularly asthma) over24 hours or more.

[0083] Topical preparations may be administered by one or moreapplications per day to the affected area; over skin areas occlusivedressings may advantageously be used. Continuous or prolonged deliverymay be achieved by an adhesive reservoir system.

[0084] For internal administration the compounds according to theinvention may, for example, be formulated in conventional manner fororal, parenteral or rectal administration. Formulations for oraladministration include syrups, elixirs, powders, granules, tablets andcapsules which typically contain conventional excipients such as bindingagents, fillers, lubricants, disintegrants, wetting agents, suspendingagents, emulsifying agents, preservatives, buffer salts, flavouring,colouring and/or sweetening agents as appropriate. Dosage unit formsare, however, preferred as described below.

[0085] Preferred forms of preparation for internal administration aredosage unit forms i.e. tablets and capsules. Such dosage unit formscontain from 0.1 mg to 20 mg preferably from 2.5 to 10 mg of thecompounds of the invention.

[0086] The compounds according to the invention may in general may begiven by internal administration in cases where systemic adreno-corticaltherapy is indicated.

[0087] In general terms preparations, for internal administration maycontain from 0.05 to 10% of the active ingredient dependent upon thetype of preparation involved. The daily dose may vary from 0.1 mg to 60mg, e.g. 5-30 mg, dependent on the condition being treated, and theduration of treatment desired.

[0088] Slow release or enteric coated formulations may be advantageous,particularly for the treatment of inflammatory bowel disorders.

[0089] The pharmaceutical compositions according to the invention mayalso be used in combination with another therapeutically active agent,for example, a β₂ adrenoreceptor agonist, an anti-histamine or ananti-allergic. The invention thus provides, in a further aspect, acombination comprising a compound of formula (I) or a physiologicallyacceptable salt or solvate thereof together with another therapeuticallyactive agent, for example, a β₂-adrenoreceptor agonist, ananti-histamine or an anti-allergic.

[0090] Examples of β₂-adrenoreceptor agonists include salmeterol (eg asracemate or a single enantiomer such as the R-enantiomer), salbutamol,formoterol, salmefamol, fenoterol or terbutaline and salts thereof, forexample the xinafoate salt of salmeterol, the sulphate salt or free baseof salbutamol or the fumarate salt of formoterol. Pharmaceuticalcompositions employing combinations with long-acting β₂-adrenoreceptoragonists (eg salmeterol and salts thereof) are particularly preferred,especially those which have a therapeutic effect (eg in the treatment ofasthma or COPD, particularly asthma) over 24 hours or more.

[0091] Since the compound of formula (I) is long-acting, preferably thecomposition comprising the compound of formula (I) and the long-actingβ₂-adrenoreceptor agonists will be delivered once-per-day and the doseof each will be selected so that the composition has a therapeuticeffect in the treatment of respiratory disorders effect (eg in thetreatment of asthma or COPD, particularly asthma) over 24 hours or more.

[0092] Examples of anti-histamines include methapyrilene or loratadine.

[0093] Other suitable combinations include, for example, otheranti-inflammatory agents eg. NSAIDs (eg. sodium cromoglycate, nedocromilsodium, PDE4 inhibitors, leukotriene antagonists, iNOS inhibitors,tryptase and elastase inhibitors, beta-2 integrin antagonists andadenosine 2a agonists)) or antiinfective agents (eg. antibiotics,antivirals).

[0094] Of particular interest is use of the compounds of formula (I) incombination with a phosphodiesterase 4 (PDE4) inhibitor. ThePDE4-specific inhibitor useful in this aspect of the invention may beany compound that is known to inhibit the PDE4 enzyme or which isdiscovered to act as a PDE4 inhibitor, and which are only PDE4inhibitors, not compounds which inhibit other members of the PDE familyas well as PDE4. Generally it is preferred to use a PDE4 inhibitor whichhas an IC₅₀ ratio of about 0.1 or greater as regards the IC₅₀ for thePDE4 catalytic form which binds rolipram with a high affinity divided bythe IC₅₀ for the form which binds rolipram with a low affinity. For thepurposes of this disclosure, the cAMP catalytic site which binds R and Srolipram with a low affinity is denominated the “low affinity” bindingsite (LPDE 4) and the other form of this catalytic site which bindsrolipram with a high affinity is denominated the “high affinity” bindingsite (HPDE 4). This term “HPDE4” should not be confused with the term“hPDE4” which is used to denote human PDE4. Initial experiments wereconducted to establish and validate a [³H]-rolipram binding assay.Details of this work are given in the Binding Assays described in detailbelow.

[0095] The preferred PDE4 inhibitors of use in this invention will bethose compounds which have a salutary therapeutic ratio, i.e., compoundswhich preferentially inhibit cAMP catalytic activity where the enzyme isin the form that binds rolipram with a low affinity, thereby reducingthe side effects which apparently are linked to inhibiting the formwhich binds rolipram with a high affinity. Another way to state this isthat the preferred compounds will have an IC₅₀ ratio of about 0.1 orgreater as regards the IC₅₀ for the PDE4 catalytic form which bindsrolipram with a high affinity divided by the IC₅₀ for the form whichbinds rolipram with a low affinity.

[0096] A further refinement of this standard is that of one wherein thePDE4 inhibitor has an IC₅₀ ratio of about 0.1 or greater; said ratio isthe ratio of the IC₅₀ value for competing with the binding of 1 μM of[³H]R-rolipram to a form of PDE4 which binds rolipram with a highaffinity over the IC₅₀ value for inhibiting the PDE4 catalytic activityof a form which binds rolipram with a low affinity using 1 μM[³H]-cAMPas the substrate.

[0097] Examples of useful PDE4 inhibitors are:

[0098](R)-(+)-1-(4-bromobenzyl)-4-[(3-cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidone;

[0099](R)-(+)-1-(4-bromobenzyl)-4-[(3-cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidone;

[0100]3-(cyclopentyloxy-4-methoxyphenyl)-1-(4-N′-[N2-cyano-S-methyl-isothioureido]benzyl)-2-pyrrolidone;

[0101] cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid];

[0102]cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];

[0103] (R)-(+)-ethyl[4-(3-cyclopentyloxy-4-methoxyphenyl)pyrrolidine-2-ylidene]acetate; and

[0104]

[0105] (S)-(−)-ethyl[4-(3-cyclopentyloxy-4-methoxyphenyl)pyrrolidine-2-ylidene]acetate.

[0106] Most preferred are those PDE4 inhibitors which have an IC₅₀ ratioof greater than 0.5, and particularly those compounds having a ratio ofgreater than 1.0. Preferred compounds are cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-oneandcis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];these are examples of compounds which bind preferentially to the lowaffinity binding site and which have an IC₅₀ ratio of 0.1 or greater.

[0107] Other compounds of interest include:

[0108] Compounds set out in U.S. Pat. No. 5,552,438 issued Sep. 3, 1996;this patent and the compounds it discloses are incorporated herein infull by reference. The compound of particular interest, which isdisclosed in U.S. Pat. No. 5,552,438, iscis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid (also known as cilomalast) and its salts, esters, pro-drugs orphysical forms;

[0109] AWD-12-281 from Astra (Hofgen, N. et al. 15th EFMC Int Symp MedChem (Sept 6-10, Edinburgh) 1998, Abst P.98); a 9-benzyladeninederivative nominated NCS-613 (INSERM); D-4418 from Chiroscience andSchering-Plough; a benzodiazepine PDE4 inhibitor identified as CI-1018(PD-168787; Parke-Davis/Warner-Lambert); a benzodioxole derivative KyowaHakko disclosed in WO 9916766; V-11294A from Napp (Landells, L. J. etal. Eur Resp J [Annu Cong Eur Resp Soc (Sept 19-23, Geneva) 1998] 1998,12(Suppl. 28): Abst P2393); roflumilast (CAS reference No 162401-32-3)and a pthalazinone (WO 9947505) from Byk-Gulden; or a compoundidentified as T-440 (Tanabe Seiyaku; Fuji, K. et al. J Pharmacol ExpTher,1998, 284(1): 162).

[0110] Phosphodiesterase and Rolipram Binding Assays

[0111] Assay Method 1A

[0112] Isolated human monocyte PDE4 and hrPDE (human recombinant PDE4)was determined to exist primarily in the low affinity form. Hence, theactivity of test compounds against the low affinity form of PDE4 can beassessed using standard assays for PDE4 catalytic activity employing 1μM [³H]cAMP as a substrate (Torphy et al., J. of Biol. Chem., Vol. 267,No. 3 pp1798-1804, 1992). Rat brain high speed supernatants were used asa source of protein and both enantiomers of [³H]-rolipram were preparedto a specific activity of 25.6 Ci/mmol. Standard assay conditions weremodified from the published procedure to be identical to the PDE assayconditions, except for the last of the cAMP: 50 mM Tris HCl (pH 7.5), 5mM MgCl₂, 50 μM 5′-AMP and 1 nM of [³H]-rolipram (Torphy et al., J. ofBiol. Chem., Vol. 267, No. 3 pp1798-1804, 1992). The assay was run for 1hour at 30° C. The reaction was terminated and bound ligand wasseparated from free ligand using a Brandel cell harvester. Competitionfor the high affinity binding site was assessed under conditions thatwere identical to those used for measuring low affinity PDE activity,expect that [³H]-cAMP was not present.

[0113] Assay Method 1B

[0114] Measurement of Phosphodiesterase Activity

[0115] PDE activity was assayed using a [³H]cAMP SPA or [³H]cGMP SPAenzyme assay as described by the supplier (Amersham Life Sciences). Thereactions were conducted in 96-well plates at room temperature, in 0.1ml of reaction buffer containing (final concentrations): 50 mM Tris-HCl,pH 7.5, 8.3 mM MgCl₂, 1.7 mM EGTA, [³H]cAMP or [³H] cGMP (approximately2000 dpm/pmol), enzyme and various concentrations of the inhibitors. Theassay was allowed to proceed for 1 hr and was terminated by adding 50 μLof SPA yttrium silicate beads in the presence of zinc sulfate. Theplates were shaken and allowed to stand at room temperature for 20 min.Radiolabeled product formation was assessed by scintillationspectrometry.

[0116] [³H]R-rolipram binding assay

[0117] The [³H]R-rolipram binding assay was performed by modification ofthe method of Schneider and co-workers, see Nicholson, et al., TrendsPharmacol. Sci., Vol. 12, pp.19-27 (1991) and McHale et al., Mol.Pharmacol., Vol. 39, 109-113 (1991). R-Rolipram binds to the catalyticsite of PDE4 see Torphy et al., Mol. Pharmacol., Vol. 39, pp. 376-384(1991). Consequently, competition for [³H]R-rolipram binding provides anindependent confirmation of the PDE4 inhibitor potencies of unlabeledcompetitors. The assay was performed at 30° C. for 1 hr in 0.5 μl buffercontaining (final concentrations): 50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂,0.05% bovine serum albumin, 2 nM [³H]R-rolipram (5.7×10⁴ dpm/pmol) andvarious concentrations of non-radiolabeled inhibitors. The reaction wasstopped by the addition of 2.5 ml of ice-cold reaction buffer (without[³H]-R-rolipram) and rapid vacuum filtration (Brandel Cell Harvester)through Whatman GF/B filters that had been soaked in 0.3%polyethylenimine. The filters were washed with an additional 7.5 ml ofcold buffer, dried, and counted via liquid scintillation spectrometry.

[0118] The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a physiologically acceptablesalt or solvate thereof together with a PDE4 inhibitor. The combinationreferred to above may conveniently be presented for use in the form of apharmaceutical formulation and thus pharmaceutical formulationscomprising a combination as defined above together with apharmaceutically acceptable diluent or carrier represent a furtheraspect of the invention.

[0119] The compound according to the invention in combination withanother therapeutically active ingredient as described above may beformulated for administration in any convenient way, and the inventiontherefore also includes within its scope pharmaceutical compositionscomprising the compound of formula (I) or a physiologically acceptablesolvate thereof in combination with another therapeutically activeingredient together, if desirable, in admixture with one or morephysiologically acceptable diluents or carriers. The preferred route ofadministration for inflammatory disorders of the respiratory tract willgenerally be administration by inhalation.

[0120] The individual compounds of such combinations may be administeredeither sequentially in separate pharmaceutical compositions as well assimultaneously in combined pharmaceutical formulations. Appropriatedoses of known therapeutic agents will be readily appreciated by thoseskilled in the art.

[0121] The compounds of formula (I) and salts and solvates thereof maybe prepared by the methodology described hereinafter, constituting afurther aspect of this invention.

[0122] A process according to the invention for preparing a compound offormula (I) or a salt or solvate thereof comprises alkylation of athioacid of formula (II)

[0123] wherein R², R³, R⁴, R⁵ and

[0124] are as defined above, or a salt thereof.

[0125] In this process the compound of formula (II) may be reacted witha compound of formula R₁-L wherein L represents a leaving group such ashalogen atom or a tosyl or mesyl group or the like, for example, anappropriate alkyl orhaloalkyl halide under standard conditions.

[0126] Compounds of formula (II) may conveniently be employed as saltswhen such salts may be prepared in crystalline form.

[0127] When R₁ represents fluoromethyl, the preferred haloalkyl halidereagent is bromofluoromethane.

[0128] In a preferred process for preparing a compound of formula (I), acompound of formula (II) or a salt thereof may be treated withbromofluoromethane optionally in the presence of a phase transfercatalyst and optionally in the presence of an added base. A preferredsolvent is methylacetate, or more preferably ethylacetate, optionally inthe presence of water. The presence of water improves solubility of bothstarting material and product and the use of a phase transfer catalystresults in an increase in rate of reaction. Examples of phase transfercatalysts that may be employed include (but are not restricted to)tetrabutylammonium bromide, tetrabutylammonium chloride,benzyltributylammonium bromide, benzyltributylammonium chloride,benzyltriethylammonium bromide, methyltributylammonium chloride andmethyltrioctylammonium chloride. THF may also advantageously be employedas solvent for the phase transfer catalyst.

[0129] Compounds of formula (II) may be prepared from the corresponding17α-hydroxyl derivative of formula (III):

[0130] or a salt thereof

[0131] wherein R³, R⁴, R5 and

[0132] are as defined above, using for example, the methodologydescribed by G. H. Phillipps et al., (1994) Journal of MedicinalChemistry, 37, 3717-3729. For example the step typically comprises theaddition of a reagent suitable for performing the esterification to theester such as an aryl or heteroarylcarbonyl halide eg. 2-furanoylchloride in the presence of a mild base eg. triethylamine. Generally thearyl or heteroarylcarbonyl halide would be employed in at least 2 timesmolar quantity relative to the compound of formula (III). The secondmole of aryl or heteroarylcarbonyl halide tends to react with thethioacid moiety in the compound of formula (III) and would need to beremoved by reaction with an amine such as diethylamine.

[0133] Compounds of formula (III) may be prepared in accordance withprocedures described in GB 2088877B.

[0134] Compounds of formula (III) wherein R₃ represents methyl in the αconfiguration,

[0135] represents a double bond and R₄ and R₅ represent F may also beprepared by a process comprising the following steps:

[0136] Step (a) comprises oxidation of a solution containing thecompound of formula (V). Preferably, step (a) will be performed in thepresence of a solvent comprising methanol, water, tetrahydrofuran,dioxan or diethylene glygol dimethylether. For example, so as to enhanceyield and throughput, preferred solvents are methanol, water ortetrahydrofuran, and more preferably are water or tetrahydrofuran,especially water and tetrahydrofuran as solvent. Dioxan and diethyleneglygol dimethylether are also preferred solvents which may optionally(and preferably) be employed together with water. Preferably, thesolvent will be present in an amount of between 3 and 10 vol relative tothe amount of the starting material (1 wt.), more preferably between 4and 6 vol., especially 5 vol. Preferably the oxidising agent is presentin an amount of 1-9 molar equivalents relative to the amount of thestarting material. For example, when a 50% w/w aqueous solution ofperiodic acid is employed, the oxidising agent may be present in anamount of between 1.1 and 10 wt. relative to the amount of the startingmaterial (1 wt.), more preferably between 1.1 and 3 wt., especially 1.3wt. Preferably, the oxidation step will comprise the use of a chemicaloxidising agent. More preferably, the oxidising agent will be periodicacid or iodic acid or a salt thereof. Most preferably, the oxidisingagent will be periodic acid or sodium periodate, especially periodicacid. Alternatively (or in addition), it will also be appreciated thatthe oxidation step may comprise any suitable oxidation reaction, eg. onewhich utilises air and/or oxygen. When the oxidation reaction utilisesair and/or oxygen, the solvent used in said reaction will preferably bemethanol. Preferably, step (a) will involve incubating the reagents atroom temperature or a little warmer, say around 25° C. eg for 2 hours.The compound of formula (I) may be isolated by recrystallisation fromthe reaction mixture by addition of an anti-solvent. A suitableanti-solvent for compound of formula (I) is water. Surprisingly we havediscovered that it is highly desirable to control the conditions underwhich the compound of formula (IV) is precipitated by addition ofanti-solvent eg water. When the recrystallisation is performed usingchilled water (eg water/ice mixture at a temperature of 0-5° C.)although better anti-solvent properties may be expected we have foundthat the crystalline product produced is very voluminous, resembles asoft gel and is very difficult to filter. Without being limited bytheory we believe that this low density product contains a large amountof solvated solvent within the crystal lattice By contrast whenconditions of around 10° C. or higher are used (eg around ambienttemperature) a granular product of a sand like consistency which is veryeasily filtered is produced. Under these conditions, crystallisationtypically commences after around 1 hour and is typically completedwithin a few hours (eg 2 hours). Without being limited by theory webelieve that this granular product contains little or no of solvatedsolvent within the crystal lattice.

[0137] Step (b) will typically comprise the addition of a reagentsuitable for converting a carboxylic acid to a carbothioic acid eg.using hydrogen sulphide gas together with a suitable coupling agent eg.carbonyldiimidazole (CDI) in the presence of a suitable solvent eg.dimethylformamide.

[0138] The aforementioned methodology may be adapted for the preparationof other compounds of formula (III).

[0139] An alternative process for preparing certain compounds of formula(II) comprises treating a compound of formula (X) with a reagentsuitable for converting a carboxylic acid to a carbothioic acid eg usinghydrogen sulphide gas together with a suitable coupling agent such asCDI in the presence of a suitable solvent eg DMF. Compounds of formula(X) may be prepared by methodology analogous to that described herein.Other compounds of formula (II) may be prepared similarly.

[0140] An alternative process for preparing a compound of formula (I)wherein R₄ represents fluorine or a salt or solvate thereof comprisesreacting a compound of formula (VI)

[0141] with a fluorine source.

[0142] Examples of suitable sources of fluorine include fluoride (egsodium fluoride) or, more preferably, HF. The preferred reagent isaqueous HF. A solvent such as THF or DMF may be employed.

[0143] A compound of formula (VI) may be prepared by a processcomprising

[0144] (a) alkylating a compound of formula (VII)

[0145] or a salt thereof;

[0146] (b) reacting a compound of formula (VIII)

[0147] with an epoxide forming reagent; or

[0148] (c) esterifying a compound of formula (IX)

[0149] In process (a), analogous conditions to those described above forthe conversion of a compound of formula (II) to a compound of formula(I) may be employed. Typically compound of formula (VII) will be reactedwith a compound of formula R₁-L wherein L represents a leaving group (ega halogen atom, a mesyl or tosyl group or the like) for example, anappropriate fluoromethyl halide under standard conditions. Preferably,the fluoromethyl halide reagent is bromofluoromethane.

[0150] Process (b) is preferably performed in two steps: (i) formationof a halohydrin especially a bromohydrin (eg by reaction with bromodanor equivalent reagent), followed by (ii) treatment with base such assodium hydroxide so as to effect ring closure. The product of step (i)is a compound of formula (IXA) which is a novel intermedate that may beisolated, if desired:

[0151] wherein X represents halogen, especially Br.

[0152] In process (c), a suitable reagent would be an activatedderivative of an aryl or heteroaryl carboxylic acid such as an activatedester or preferably an acid halide eg an acid chloride in the presenceof an organic base eg triethylamine. This reaction may be performed atelevated temperature eg around 60° C. or else at ambient temperature inthe presence of an acylation catalyst eg dimethylamino pyridine (DMAP).

[0153] Compounds of formula (VII) may be prepared by a processcomprising esterification of a compound of formula (XI)

[0154] Analogous conditions to those described above for the conversionof a compound of formula (III) to a compound of formula (II) may beemployed. For example, a suitable reagent would be an activatedderivative of an aryl or heteroaryl carboxylic acid such as an activatedester or preferably an acid halide eg acid chloride in the presence ofan organic base eg triethylamine. Certain compounds of formula (XI) areknown (J Labelled Compd Radiopharm (1997) 39(7) 567-584) and others maybe prepared by analogous methods.

[0155] A compound of formula (VIII) may be prepared by a processcomprising

[0156] (a) alkylating a compound of formula (XII)

[0157] or a salt thereof; or

[0158] (b) esterifying a compound of formula (XIII)

[0159] In process (a), analogous conditions to those described above forthe conversion of a compound of formula (II) to a compound of formula(I) may be employed. Typically compound of formula (XII) will be reactedwith a compound of formula R₁-L wherein L represents a leaving group (ega halogen atom, a mesyl or tosyl group or the like) for example, anappropriate fluoromethyl halide under standard conditions. Preferably,the fluoromethyl halide reagent is bromofluoromethane.

[0160] In process (b), analogous conditions to those employed above forthe conversion of a compound of formula (IX) to a compound of formula(VI) may be employed. For example, a suitable reagent would be anactivated derivative of an aryl or heteroaryl carboxylic acid such as anactivated ester or preferably an acid halide eg acid chloride in thepresence of an organic base eg triethylamine.

[0161] Compounds of formula (IX) and (XIII) may be prepared byalkylating the corresponding thioacids (XI) and (XIV) (defined below)using methodology analogous to that already described (eg by reactionwith a compound of formula FCH₂L wherein L represents a leaving group(eg a halogen atom, a mesyl or tosyl group or the like) for example, anappropriate fluoromethyl halide under standard conditions. Preferably,the fluoromethyl halide reagent is bromofluoromethane. Thethioacids (XI)are either known compounds (J Labelled Compd Radiopharm (1997) 39(7)567-584) or may be prepared by analogous methods.

[0162] Compounds of formula (XII) may be prepared by a processcomprising esterifying a compound of formula (XIV):

[0163] or a salt thereof.

[0164] This process may be performed using methodology analogous to thatalready described. For example, a suitable reagent would be an activatedderivative of an aryl or heteroaryl carboxylic acid such as an activatedester or preferably an acid halide eg and acid chloride in the presenceof an organic base eg triethylamine.

[0165] Compounds of formula (XIV) may be prepared from the correspondingcarboxylic acid eg by a process analogous to that described above forthe conversion of a compound of formula (IV) to a compound of formula(III). The aforesaid the corresponding carboxylic acid is either known(Upjohn WO90/15816) or may be prepared by conventional methods.

[0166] A further alternative process for preparing a compound of formula(I) or a salt or solvate thereof comprises deprotecting or unmasking acompound of formula (I) in which the 11-β-hydroxy group is protected ormasked. A first such process comprises deprotecting a compound offormula (XV)

[0167] wherein P represents a hydroxy protecting group.

[0168] Examples of hydroxy protecting groups P are described inProtective Groups in Organic Chemistry Ed J F W McOmie (Plenum Press1973) or Protective Groups in Organic Synthesis by Theodora W Green(John Wiley and Sons, 1991).

[0169] Examples of suitable hydroxy protecting groups P include groupsselected from carbonate, alkyl (eg t-butyl or methoxymethyl), aralkyl(eg benzyl, p-nitrobenzyl, diphenylmethyl or triphenylmethyl),heterocyclic groups such as tetrahydropyranyl, acyl (eg acetyl orbenzyl) and silyl groups such as trialkylsilyl (egt-butyldimethylsilyl). The hydroxy protecting groups may be removed byconventional techniques. Thus, for example, carbonate may be removed bytreatment with base and alkyl, silyl, acyl and heterocyclic groups maybe removed by solvolysis eg by hydrolysis under acid or basicconditions. Aralkyl groups such as triphenylmethyl may similarly beremoved by solvolysis eg by hydrolysis under acidic conditions. Aralkylgroups such as benzyl or p-nitrobenzyl may be cleaved by hydrogenolysisin the presence of a Noble metal catalyst such as palladium on charcoal.p-Nitrobenzyl may also be cleaved by photolysis.

[0170] The 11-β-hydroxy group may be masked as a carbonyl group. Thus asecond such process comprises reduction of a compound of formula (XVI)

[0171] Reduction to the compound of formula (I) may be achieved eg bytreatment with a hydride reducing agent such as borohydride eg sodiumborohydride

[0172] The 11-ketone (XVI) may also be masked. Examples of maskedderivatives of compound of formula (XVI) include (i) ketal derivativeseg ketals formed by treatment of the compound of formula (XVI) with analcohol eg methanol, ethanol or ethan-1,2-diol, (ii) dithioketalderivatives eg dithioketals formed by treatment of a compound of formula(XVI) with a thiol eg methanethiol, ethanethiol or ethan-1,2-dithiol,(iii) monothioketal derivatives eg monothioketals formed by treatment ofa compound of formula (XVI) with eg 1-hydroxy-ethane-2-thiol, (iv)derivatives formed by treatment of a compound of formula (XVI) with analcoholamine eg ephedrine, (v) imines formed by treatment of a compoundof formula (XVI) with amines, (vi)oximes formed by treatment ofcompounds of formula (XVI) with hydroxylamines. We claims suchderivatives of compounds of formula (XVI) as an aspect of the invention.

[0173] These masked derivatives may be converted back to the ketone byconventional means eg ketals, imines and oximes are converted tocarbonyl by treatment with dilute acid and dithioketals are converted tothe ketone by a variety of methods as described by P. C. Bulman Page etal (1989), Tetrahedron, 45, 7643-7677 and references therein.

[0174] Compounds of formula (XV) may be prepared by a process comprising

[0175] (a) alkylating a compound of formula (XVII)

[0176] or a salt thereof wherein P represents a hydroxy protectinggroup; or

[0177] (b) esterifying a compound of formula (XVIII)

[0178] In step (a), analogous conditions to those described above forthe conversion of a compound of formula (II) to a compound of formula(I) may be employed. Typically compound of formula (XVII) will bereacted with a compound of formula R₁-L wherein L represents a leavinggroup (eg a halogen atom, a mesyl or tosyl group or the like) forexample, an appropriate fluoromethyl halide under standard conditions.Preferably, the fluoromethyl halide reagent is bromofluoromethane.

[0179] In step (b), analogous conditions to those employed above for theconversion of a compound of formula (IX) to a compound of formula (VI)may be employed. For example, a suitable reagent would be an activatedderivative of an aryl or heteroaryl carboxylic acid such as an activatedester or preferably an acid halide eg acid chloride in the presence ofan organic base eg triethylamine.

[0180] Compound of formula (XVIII) may be prepared by alkylating thecorresponding thioacid using methodology analogous to that alreadydescribed (eg by reaction with a compound of formula R₁ -L wherein Lrepresents a leaving group (eg a halogen atom, a mesyl or tosyl group orthe like) for example, an appropriate fluoromethyl halide under standardconditions. When R₁ represents —CH₂F, preferably, the fluoromethylhalide reagent is bromofluoromethane. The corresponding thioacids areknown compounds or may be prepared by known methods. Compound of formula(XVIII) may alternatively be prepared by protection of the correspondinghydroxy derivative.

[0181] Compound of formula (XVII) may be prepared by a processcomprising esterifying a compound of formula (XIX)

[0182] or a salt thereof wherein P represents a hydroxy protectinggroup.

[0183] This process may be performed using methodology analogous to thatalready described. For example, a suitable reagent would be an activatedderivative of an aryl or heteroaryl carboxylic acid such as an activatedester or preferably an acid halide eg acid chloride in the presence ofan organic base eg triethylamine.

[0184] Compounds of formula (XIX) may be prepared by protecting thecorresponding hydroxy derivative, having first protected the thioacidwhich would need to be subsequently deprotected. The correspondinghydroxy derivatives are known compounds or may be prepared by knownmethods.

[0185] Compounds of formula (XVI) may be prepared by a processcomprising

[0186] (a) alkylating a compound of formula (XX)

[0187] or a salt thereof or a derivative wherein the 11-carbonyl groupis masked; or

[0188] (b) esterifying a compound of formula (XXI)

[0189] or a derivative wherein the 11-carbonyl group is masked.

[0190] In step (a), analogous conditions to those described above forthe conversion of a compound of formula (III) to a compound of formula(II) may be employed. Typically compound of formula (XX) will be reactedwith a compound of formula R₁-L wherein L represents a leaving group (ega halogen atom, a mesyl or tosyl group or the like) for example, anappropriate fluoromethyl halide under standard conditions. Preferably,the fluoromethyl halide reagent is bromofluoromethane.

[0191] In step (b), analogous conditions to those employed above for theconversion of a compound of formula (IX) to a compound of formula (VI)may be employed. For example, a suitable reagent would be an activatedderivative of an aryl or heteroaryl carboxylic acid such as an activatedester or preferably an acid halide eg acid chloride in the presence ofan organic base eg triethylamine.

[0192] Compound of formula (XXI) or a derivative thereof wherein the11-ketone group is masked may be prepared by alkylating thecorresponding thioacid using methodology analogous to that alreadydescribed (eg by reaction with a compound of formula R₁-L wherein Lrepresents a leaving group (eg a halogen atom, a mesyl or tosyl group orthe like) for example, an appropriate fluoromethyl halide under standardconditions. Preferably, the fluoromethyl halide reagent isbromofluoromethane. The corresponding thioacids are known compounds.

[0193] Compound of formula (XX) may be prepared by a process comprisingesterifying a compound of formula (XXII)

[0194] or a salt thereof or a derivative thereof wherein the 11-ketonegroup is masked.

[0195] This process may be performed using methodology analogous to thatalready described. For example, a suitable reagent would be an activatedderivative of an aryl or heteroaryl carboxylic acid such as an activatedester or preferably an acid halide eg acid chloride in the presence ofan organic base eg triethylamine.

[0196] Compounds of formula (XXII) and derivatives thereof wherein the11-ketone is masked may be prepared by oxidation of the correspondinghydroxy derivative (IV) (or analogue thereof) followed by masking of theketone and subsequent conversion of the carboxylic acid group to thethioacid (see eg conversion of compounds of formula (IV) to (III)).

[0197] A further alternative process for the preparation of compounds offormula (I) wherein R₁ represents —CH₂F comprises reaction of a compoundof formula (XXIII)

[0198] wherein L represents a leaving group (eg halide other thanfluoride such as chloride, iodide or a sulphonate ester such mesylate,tosylate, triflate) with a fluorine source.

[0199] Preferably the fluorine source is fluoride ion eg KF. Furtherdetails for this conversion may be obtained by reference to G. H.Phillipps et al., (1994) Journal of Medicinal Chemistry, 37, 3717-3729or J Labelled Compd Radiopharm (1997) 39(7) 567-584).

[0200] Some compounds of formula (XXIII) are compounds of formula (I).Compounds of formula (XXIII) may be prepared by methods analogous tothose described herein. Corresponding novel intermediates of formula(VI), (VII), (IX), (1×A), (XV) and (XVI) wherein the —CH₂F moiety isreplaced with a —CH₂L moiety (wherein L represents a leaving group otherthan fluorine) are claimed as an aspect of the invention.

[0201] A further alternative process for the preparation of compounds offormula (I) or a solvate thereof comprises deprotection or unmasking ofa derivative of a compound of formula (I) in which the 3-carbonyl groupis protected or masked.

[0202] The 3-carbonyl group may be masked in a manner analogous to thatdescribed above in relation to masking of the 11-carbonyl position. Thusthe 3-carbonyl may be masked eg as a ketal, monothioketal, dithioketal,derivative with an alcoholamine, oxime or imine. The carbonyl group maybe recovered by conventional means eg ketals are converted to carbonylby treatment with dilute acid and dithioketals are converted to theketone by a variety of methods as described by P. C. Bulman Page et al(1989), Tetrahedron, 45, 7643-7677 and references therein.

[0203] Certain intermediate compounds are new and we provide these,together where appropriate with their salts and solvates, as an aspectof the invention.

[0204] The advantages of the compounds of formula (I) and/or itssolvates may include the fact that the substance appears to demonstrateexcellent anti-inflammatory properties, with predictable pharmacokineticand pharmacodynamic behaviour, with an attractive side-effect profile,long duration of action, and is compatible with a convenient regime oftreatment in human patients, in particular being amendable to once-perday dosing. The advantages may be appreciated in particular when thecompound of formula (I) and/or its solvates are employed in combinatoinwith a the long-acting β₂-adrenoreceptor agonist. Further advantages mayinclude the fact that the substance has desirable physical and chemicalproperties which allow for ready manufacture and storage.

[0205] The following non-limiting Examples illustrate the invention:

EXAMPLES

[0206] General

[0207]¹H-nmr spectra were recorded at 400 MHz and the chemical shiftsare expressed in ppm relative to tetramethylsilane. The followingabbreviations are used to describe the multiplicities of the signals: s(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd(doublet of doublets), dt (doublet of triplets) and b (broad). Biotagerefers to prepacked silica gel cartridges containing KP-Sil run on flash12i chromatography module. LCMS was conducted on a Supelcosil LCABZ+PLUScolumn (3.3 cm×4.6 mm ID) eluting with 0.1% HCO₂H and 0.01 M ammoniumacetate in water (solvent A), and 0.05% HCO₂H 5% water in acetonitrile(solvent B), using the following elution gradient 0-0.7 min 0%B, 0.7-4.2min 100%B, 4.2-5.3 min 0%B, 5.3-5.5 min 0%B at a flow rate of 3 ml/min.The mass spectra were recorded on a Fisons VG Platform spectrometerusing electrospray positive and negative mode (ES+ve and ES−ve).

[0208] Intermediates

[0209] Intermediate 1:6α,9α-Difluoro-17α[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicAcid

[0210] A solution of 6α, 9α-difluoro-11β,17α-dihydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid(prepared in accordance with the procedure described in GB 2088877B) (18g, 43.64 mmol) in anhydrous dichloromethane (200 ml) and triethylamine(15.94 ml, 114 mmol) was treated at <5° C. with a solution of 2-furoylchloride (11.24 ml, 114 mmol) in anhydrous dichloromethane (100 ml) overapproximately 40 min. The solution was stirred at <5° C. for 30 min. Theresulting solid was collected by filtration, washed successively with3.5% aqueous sodium hydrogen carbonate solution, water, 1 M hydrochloricacid, and water and dried in vacuo at 60° C. to give a cream colouredsolid. The dichloromethane filtrate was washed successively with 3.5%sodium hydrogen carbonate solution, water, 1 M hydrochloric acid, water,dried (Na₂SO₄) and evaporated to give a cream coloured solid which wascombined with that isolated above. The combined solids (26.9 g) weresuspended in acetone (450 ml) and stirred. Diethylamine (16.8 ml, 162mmol) was added and the mixture stirred at room temperature for 4.5 h.The mixture was concentrated and the precipitate collected by filtrationand washed with a little acetone. The washings and filtrate werecombined, concentrated and loaded onto a silica gel Biotage column whichwas eluted with 24:1 chloroform:methanol. Fractions which contained themore polar component were combined and evaporated to give a creamcoloured solid. This was combined with the solid isolated above anddried in vacuo to give a pale beige coloured solid (19.7 g). This wasdissolved in warm water, the pH adjusted to 2 with concentratedhydrochloric acid and the mixture extracted with ethyl acetate. Theorganic extract was dried (Na₂SO₄) and evaporated to give, after dryingat 50° C., the title compound as a cream coloured solid (18.081 g, 82%):LCMS retention time 3.88 min, m/z 507 MH⁺, NMR δ (CDCl₃) includes 7.61(1H, m), 7.18-7.12 (2H, m), 6.52 (1H, dd, J 4, 2 Hz), 6.46 (1H, s), 6.41(1H, dd, J 10, 2 Hz), 5.47 and 5.35 (1H, 2 m), 4.47 (1H, bd, J 9 Hz),3.37 (1H, m), 1.55 (3H, s), 1.21 (3H, s), 1.06 (3H, d, J 7 Hz).

[0211] The following intermediates were prepared using a methodanalogous to that described for Intermediate 1:

[0212] Intermediate 2:6α,9α-Difluoro-17α[(3-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0213] LCMS retention time 3.75 min, m/z 507 MH⁺.

[0214] Intermediate 3:6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(2-thienylcarbonyl)oxy]-androsta-1.4-diene-17β-carbothioicacid

[0215] LCMS retention time 3.93 min, m/z 523 MH⁺.

[0216] Intermediate 4:6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(3-thienylcarbonyl)oxy]-androsta-1,4-diene-17β-carbothioicacid

[0217] LCMS retention time 3.95 min, m/z 523 MH⁺.

[0218] Intermediate 5:17α-(Benzoyl)oxy-6α,9αa-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid

[0219] LCMS retention time 4.02 min, m/z 517 MH⁺.

[0220] Intermediate 6:9α-Fluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16β-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0221] Intermediate 6was prepared from11β,17α-dihydroxy-9α-fluoro-16β-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid (prepared in accordance with theprocedure described in Phillips et al, (1994) J. Med. Chem. 37,3717-3729). LCMS retention time 3.61 min, m/z 489 MH⁺.

[0222] Intermediate 7:6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(5-methylthiophene-2-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0223] LCMS retention time 4.01 min, m/z 537 MH⁺

[0224] Intermediate 8:6α,9α-Difluoro-11β-hydroxy-17α-[(isoxazole-5-carbonyl)oxy]-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0225] LCMS retention time 3.69 min, m/z 508 MH⁺

[0226] Intermediate 9:17α-[(5-Chlorothiophene-2-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0227] LCMS retention time 4.36 min, m/z 557/559 MH⁺

[0228] Intermediate 10:6α,9α-Difluoro-17α-[(3,5-dimethylisoxazole-4-carbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0229] LCMS retention time 3.71 min, m/z 536 MH⁺

[0230] Intermediate 11:17α-[(5-Chloro-4-methoxy-thiophene-3-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0231] LCMS retention time 4.18 min, m/z 587/589 MH⁺

[0232] Intermediate 12:6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,2,3-thiadiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0233] LCMS retention time 4.06 min, m/z 539 MH⁺

[0234] Intermediate 13:17α-[(3-Bromothiophene-2-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0235] LCMS retention time 4.31 min, m/z 601/603 MH⁺

[0236] Intermediate 14:17α-[(2,5-Dichlorothiophene-3-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0237] LCMS retention time 4.59 min, m/z 591/593/595 MH⁺

[0238] Intermediate 15:17α-[(5-Bromofuran-2-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid

[0239] LCMS retention time 4.14 min, m/z 585/587 MH⁺

[0240] Intermediate 16:6α,9α-Difluoro-17α-[(2,5-dimethylfuran-3-carbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0241] LCMS retention time 4.02 min, m/z 535 MH⁺

[0242] Intermediate 17:17α-[(3-Chlorothiophene-2-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0243] LCMS retention time 4.27 min, m/z 557/559 MH⁺

[0244] Intermediate 18:6α,9α-Difluoro-11β,-hydroxy-16α-methyl-17α-[(2-methylfuran-3-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0245] LCMS retention time 3.92 min, m/z 521 MH⁺

[0246] Intermediate 19:6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(3-methylfuran-2-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0247] LCMS retention time 3.85 min, m/z 521 MH⁺

[0248] Intermediate 20:6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(5-methylisoxazole-4-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0249] LCMS retention time 3.74 min, m/z 522 MH⁺

[0250] Intermediate 21:6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(1-methyl-1H-pyrrole-2-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioic acid

[0251] LCMS retention time 3.78 min, m/z 520 MH⁺

[0252] Intermediate 22:6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(1,3-thiazole-4-carbonyl)oxy]-androsta-1,4-diene-17β-carbothioicacid

[0253] LCMS retention time 3.48 min, m/z 524 MH⁺

[0254] Intermediate 23:6α,9α-Difluoro-17α-[(2,4-dimethyl-1,3-thiazole-5-carbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0255] LCMS retention time 3.72 min, m/z 552 MH⁺

[0256] Intermediate 246α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(5-methylisoxazole-3-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0257] LCMS retention time 3.72 min, m/z 522 MH⁺

[0258] Intermediate 25:6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(3-methylisoxazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0259] LCMS retention time 3.84 min, m/z 522 MH⁺

[0260] Intermediate 26:6α,9α-Difluoro-17α-[(1,3-dimethyl-1H-pyrazole-5-carbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0261] LCMS retention time 3.72 min, m/z 535 MH⁺

[0262] Intermediate 27:6α,9α-Difluoro-11β,-hydroxy-16α-methyl-3-oxo-17α-[(1,2,3-thiadiazole-5-carbonyl)oxy]-androsta-1,4-diene-17β-carbothioicacid

[0263] LCMS retention time 3.89 min, m/z 525 MH⁺

[0264] Intermediate 28:6α,9α-Difluoro-11β-hydroxy-17α-[(isoxazole-3-carbonyl)oxy]-16α-methyl-3-oxo-androsta-1.4-diene-17β-carbothioicacid

[0265] LCMS retention time 3.78 min, m/z 508 MH⁺

[0266] Intermediate 29:6α,9α-Difluoro-11β-hydroxy-17α-[(4-methoxy-thiophene-3-carbonyl)oxy]-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0267] LCMS retention time 3.74 min, m/z 553 MH⁺

[0268] Intermediate 306α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(2-methyl-1,3-thiazole-4-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0269] LCMS retention time 3.70 min, m/z 538 MH⁺

[0270] Intermediate 31:6α,9α-Difluoro-17α-[(3-ethoxy-thiophene-2-carbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0271] LCMS retention time 3.85 min, m/z 567 MH⁺

[0272] Intermediate 32:6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(1,2,3-thiadiazole-4-carbonyl)oxy]-androsta-1,4-diene-17β-carbothioicacid

[0273] LCMS retention time 3.70 min, m/z 526 MH⁺

[0274] Intermediate 33:6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(1H-pyrrole-2-carbonyl)oxy]-androsta-1,4-diene-17β-carbothioicacid

[0275] LCMS retention time 3.75 min, m/z 506 MH⁺

[0276] Intermediate 34:6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(1,3-thiazole-5-carbonyl)oxy]-androsta-1,4-diene-17β-carbothioicacid

[0277] LCMS retention time 4.29 min, m/z 524 MH⁺

[0278] Intermediate 35:6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(1,2,5-thiadiazole-3-carbonyl)oxy]-androsta-1,4-diene-17β-carbothioicacid

[0279] LCMS retention time 4.65 min, m/z 525 MH⁺

[0280] Intermediate 36:6α,9α-Difluoro-11β-hydroxy-17α-[(isothiazole-3-carbonyl)oxy]-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0281] LCMS retention time 4.44 min, m/z 524 MH⁺

[0282] Intermediate 37:6α,9α-Difluoro-11β-hydroxy-17α-[(isothiazole-5-carbonyl)oxy]-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0283] LCMS retention time 4.60 min, m/z 524 MH⁺

[0284] Intermediate 38:6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(3-methylthiophene-2-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0285] LCMS retention time 4.31 min, m/z 537 MH⁺

[0286] Intermediate 39:6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0287] LCMS retention time 4.19 min, m/z 538 MH⁺

[0288] Intermediate 40:17α-[(1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0289] LCMS retention time 4.33 min, m/z 549 MH⁺

[0290] Intermediate 41:6α,9α-Difluoro-17α-[(1-methyl-1H-imidazole-5-carbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid

[0291] LCMS retention time 3.91 min, m/z 521 MH⁺

EXAMPLES Example 16α,9α-Difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicAcid S-fluoromethyl Ester

[0292] A suspension of Intermediate 1 (2.5 g, 4.94 mmol) was dissolvedin anhydrous N,N-dimethylformamide (25 ml) and sodium hydrogen carbonate(465 mg, 5.53 mmol) was added. The mixture was stirred at −20° C. andbromofluoromethane (0.77 ml, 6.37 mmol) was added and the mixture wasstirred at −20° C. for 2 h. Diethylamine (2.57 ml, 24.7 mmole) was addedand the mixture stirred at −20° C. for 30 min. The mixture was added to2M hydrochloric acid (93 ml) and stirred for 30 min. Water (300 ml) wasadded and the precipitate was collected by filtration, washed with waterand dried in vacuo at 50° C. to give a white solid which wasrecrystallised from acetone/water and dried in vacuo at 50° C. to givethe title compound(2.351 g, 88%): LCMS retention time 3.66 min, m/z 539MH⁺, NMR δ (CDCl₃) includes 7.60 (1H, m), 7.18-7.11 (2H, m), 6.52 (1H,dd, J4.2 Hz), 6.46 (1H, s), 6.41 (1H, dd, J 10, 2 Hz), 5.95 and 5.82 (2Hdd, J 51, 9 Hz), 5.48 and 5.35 (1H, 2 m), 4.48 (1H, m), 3.48 (1H, m),1.55 (3H, s), 1.16 (3H, s), 1.06 (3H, d, J 7 Hz).

Example 26α,9α-Difluoro-17α-[(3-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0293] Example 2 was prepared from Intermediate 2 using a methodanalogous to that described for Example 1.

[0294] LCMS retention time 3.72 min, m/z 539 MH⁺.

Example 36α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(2-thienylcarbonyl)oxy]-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0295] Example 3 was prepared from Intermediate 3 using a methodanalogous to that described for Example 1.

[0296] LCMS retention time 3.81 min, m/z 555 MH⁺.

Example 46α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(3-thienylcarbonyl)oxy]-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0297] Example 4 was prepared from Intermediate 4 using a methodanalogous to that described for Example 1.

[0298] LCMS retention time 3.82 min, m/z 555 MH⁺.

Example 517α-(Benzoyl)oxy-6α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17-carbothioicacid S-fluoromethyl ester

[0299] Example 5 was prepared from Intermediate 5 using a methodanalogous to that described for Example 1. LCMS retention time 3.73 min,m/z 549 MH⁺.

Example 69α-Fluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16β-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0300] Example 6 was prepared from Intermediate 6 using a methodanalogous to that described for Example 1.

[0301] LCMS retention time 3.61 min, m/z 521 MH⁺.

Example 76α,9α-Difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androst-4-ene-17β-carbothioicacid S-fluoromethyl ester

[0302] A solution of Example 1 (500 mg, 0.93 mmol) and Wilkinson'scatalyst (150 mg) in a mixture of toluene (14 ml) and ethanol (7 ml) wasstirred in an atmosphere of hydrogen for 23 h. The solution wasevaporated to dryness and the residue purified by Biotage chromatographyusing ethyl acetate:petroleum ether (1:2) as eluant to give a yellowsolid (435 mg) which was recrystallised (ethyl acetate/petroleum ether)to give the title compound (364 mg, 72%).

[0303] LCMS retention time 3.57 min, m/z 541 MH⁺.

Example 86α,9α-Difluoro-11α-hydroxy-17α-[(isoxazole-5-carbonyl)oxy]-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester

[0304] Example 8 was prepared from Intermediate 8 using a methodanalogous to that described for Example 1: LCMS retention time 3.47 min,m/z 540 MH⁺

Example 917α-[(5-Chlorothiophene-2-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethylester

[0305] Example 9 was prepared from Intermediate 9 using a methodanalogous to that described for Example 1: LCMS retention time 3.89 min,m/z 589/591 MH⁺

Example 106α,9α-Difluoro-17α-[(3,5-dimethylisoxazole-4-carbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0306] Example 10 was prepared from Intermediate 10 using a methodanalogous to that described for Example 1: LCMS retention time 3.70 min,m/z 568 MH⁺

Example 1117α-[(5-Chloro-4-methoxy-thiophene-3-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acidS-fluoromethyl ester

[0307] Example 11 was prepared from Intermediate 11 using a methodanalogous to that described for Example 1: LCMS retention time 3.99 min,m/z 619/621 MH⁺

Example 126α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,2,3-thiadiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0308] Example 12 was prepared from Intermediate 12 using a methodanalogous to that described for Example 1: LCMS retention time 3.74 min,m/z 571 MH⁺

Example 1317α-[(3-Bromothiophene-2-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0309] Example 13 was prepared from Intermediate 13 using a methodanalogous to that described for Example 1: LCMS retention time 3.92 min,m/z 633/635 MH⁺

Example 1417α-[(2,5-Dichlorothiophene-3-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0310] Example 14 was prepared from Intermediate 14 using a methodanalogous to that described for Example 1: LCMS retention time 4.17 min,m/z 623/625/627 MH⁺

Example 1517α-[(5-Bromofuran-2-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0311] Example 15 was prepared from Intermediate 15 using a methodanalogous to that described for Example 1: LCMS retention time 3.78 min,m/z 615/617 MH⁺

Example 166α,9α-Difluoro-17α-[(2,5-dimethylfuran-3-carbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0312] Example 16 was prepared from Intermediate 16 using a methodanalogous to that described for Example 1: LCMS retention time 3.85 min,m/z 576 MH⁺

Example 1717α-[(3-Chlorothiophene-2-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0313] Example 17 was prepared from Intermediate 17 using methodanalogous to that described for Example 1: LCMS retention time 3.88 min,m/z 589/591 MH⁺

Example 186α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(2-methylfuran-3-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0314] Example 18 was prepared from Intermediate 18 using a methodanalogous to that described for Example 1: LCMS retention time 3.74 min,m/z 553 MH⁺

Example 196α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(3-methylfuran-2-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0315] Example 19 was prepared from Intermediate 19 using a methodanalogous to that described for Example 1: LCMS retention time 3.66 min,m/z 553 MH⁺

Example 206α,9α-Difluoro-11βhydroxy-16α-methyl-17α-[(5-methylisoxazole-4-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0316] Example 20 was prepared from Intermediate 20 using a methodanalogous to that described for Example 1: LCMS retention time 3.60 min,m/z 554 MH⁺

Example 216α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(1-methyl-1H-pyrrole-2-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0317] Example 21 was prepared from Intermediate 21 using a methodanalogous to that described for Example 1: LCMS retention time 3.72 min,m/z 552 MH⁺

Example 226α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(1,3-thiazole-4-carbonyl)oxy]-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0318] Example 22 was prepared from Intermediate 22 using a methodanalogous to that described for Example 1: LCMS retention time 3.47 min,m/z 552 MH⁺

Example 236α,9α-Difluoro-17α-[(2,4-dimethyl-1,3-thiazole-5-carbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester

[0319] Example 23 was prepared from Intermediate 23 using a methodanalogous to that described for Example 1: LCMS retention time 3.51 min,m/z 584 MH⁺

Example 246α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(5-methylisoxazole-3-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0320] Example 24 was prepared from Intermediate 24 using a methodanalogous to that described for Example 1: LCMS retention time 3.65 min,m/z 554 MH⁺

Example 256α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(3-methylisoxazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0321] Example 25 was prepared from Intermediate 25 using a methodanalogous to that described for Example 1: LCMS retention time 3.52 min,m/z 554 MH⁺

Example 266α,9α-Difluoro-17α-[(1,3-dimethyl-1H-pyrazole-5-carbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0322] Example 26 was prepared from Intermediate 26 using a methodanalogous to that described for Example 1: LCMS retention time 3.52 min,m/z 567 MH⁺

Example 276α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(1,2,3-thiadiazole-5-carbonyl)oxy]-androsta-1,4-diene-17β-carbothioic acid S-fluoromethylester

[0323] Example 27 was prepared from Intermediate 27 using a methodanalogous to that described for Example 1: LCMS retention time 3.47 min,m/z 557 MH⁺

Example 286α,9α-Difluoro-11β-hydroxy-17α-[(isoxazole-3-carbonyl)oxy]-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0324] Example 28 was prepared from Intermediate 28 using a methodanalogous to that described for Example 1: LCMS retention time 3.48 min,m/z 540 MH⁺

Example 296α,9α-Difluoro-11β-hydroxy-17α-[(4-methoxy-thiophene-3-carbonyl)oxy]-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0325] Example 29 was prepared from Intermediate 29 using a methodanalogous to that described for Example 1: LCMS retention time 3.69 min,m/z 585 MH^(+Example) 306α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(2-methyl-1,3-thiazole-4-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0326] Example 30 was prepared from Intermediate 30 using a methodanalogous to that described for Example 1: LCMS retention time 3.47 min,m/z 570 MH⁺

Example 316α,9α-Difluoro-17α-[(3-ethoxy-thiophene-2-carbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethylester

[0327] Example 31 was prepared from Intermediate 31 using a methodanalogous to that described for Example 1: LCMS retention time 3.68 min,m/z 599 MH⁺

Example 326α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(1,2,3-thiadiazole-4-carbonyl)oxy]-androsta-1,4-diene-17β-carbothioic acid S-fluoromethylester

[0328] Example 32 was prepared from Intermediate 32 using a methodanalogous to that described for Example 1: LCMS retention time 3.30 min,m/z 557 MH⁺

Example 336α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(1H-pyrrole-2-carbonyl)oxy]-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0329] Example 33 was prepared from Intermediate 33 using a methodanalogous to that described for Example 1: LCMS retention time 3.42 min,m/z 528 MH⁺

Example 346α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-[(1,3-thiazole-5-carbonyl)oxy]-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0330] Example 34 was prepared from Intermediate 34 using a methodanalogous to that described for Example 1: LCMS retention time 3.44 min,m/z 556 MH⁺

Example 356α,9α-Difluoro-11β-hydroxy-16α-methyl-3-oxo-17α[(1,2,5-thiadiazole-3-carbonyl)oxy]-androsta-1,4-diene-17β-carbothioic acid S-fluoromethylester

[0331] Example 35 was prepared from Intermediate 35 using a methodanalogous to that described for Example 1: LCMS retention time 3.53 min,m/z 557 MH⁺

Example 366α,9α-Difluoro-11β-hydroxy-17α-[(isothiazole-3-carbonyl)oxy]-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0332] Example 36 was prepared from Intermediate 36 using a methodanalogous to that described for Example 1: LCMS retention time 3.51 min,m/z 556 MH⁺

Example 376α,9α-Difluoro-11β-hydroxy-17α-[(isothiazole-5-carbonyl)oxy]-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0333] Example 37 was prepared from Intermediate 37 using a methodanalogous to that described for Example 1: LCMS retention time 3.59 min,m/z 556 MH⁺

Example 386α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(5-methylthiophene-2-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0334] Example 38 was prepared from Intermediate 7 using a methodanalogous to that described for Example 1: LCMS retention time 3.78 min,m/z 569 MH⁺

Example 396α,9α-Difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-methyl ester

[0335] A suspension of Intermediate 1 (507 mg, 1 mmol) was dissolved inanhydrous N,N-dimethylformamide (5 ml) and sodium hydrogen carbonate (92mg, 1.1 mmol) was added. The mixture was stirred at 0° C. andiodomethane (0.125 ml, 2 mmol) was added and the mixture was stirred at0-5° C. for 2.5 h. Diethylamine (0.41 ml, 4 mmole) was added and themixture stirred at 5° C. for 30 min. The mixture was added to 2Mhydrochloric acid (25 ml) to give a white precipitate. Water (75 ml) wasadded and the precipitate was collected by filtration to give the titlecompound as a white solid (456 mg, 88%): LCMS retention time 3.54 min,m/z 521 MH⁺.

Example 406α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(3-methylthiophene-2-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0336] Example 40 was prepared from Intermediate 38 using a methodanalogous to that described for Example 1: LCMS retention time 3.78 min,m/z 569 MH⁺

Example 416α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0337] Example 41 was prepared from Intermediate 39 using a methodanalogous to that described for Example 1: LCMS retention time 3.51 min,m/z 570 MH⁺

Example 4217α-[(1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)oxy]-6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0338] Example 42 was prepared from Intermediate 40 using a methodanalogous to that described for Example 1: LCMS retention time 3.64 min,m/z 581 MH⁺

Example 436α,9α-Difluoro-17α-[(1-methyl-1H-imidazole-5-carbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0339] Example 43 was prepared from Intermediate 41 using a methodanalogous to that described for Example 1: LCMS retention time 3.14 min,m/z 553 MH⁺

Example 44 Dry powder composition containing6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0340] A dry powder formulation may be prepared as follows:6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl- 0.20 mg1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β- carbothioicacid S-fluoromethyl ester (prepared according to Example 41 andmicronised to a MMD of 3 μm): milled lactose (wherein not greater than85% of particles 12 mg have a MMD of 60-90 μm, and not less than 15% ofparticles have a MMD of less than 15 μm):

[0341] A peelable blister strip containing 60 blisters each filled witha formulation as just described may be prepared.

Example 45 Dry powder composition containing6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester and a long acting β₂-adrenoreceptor agonist

[0342] A dry powder formulation may be prepared as follows:6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl- 0.20 mg1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β- carbothioicacid S-fluoromethyl ester (prepared according to Example 41 andmicronised to a MMD of 3 μm): Long-acting β₂-adrenoreceptor agonist(micronised to a MMD 0.02 mg of 3 μm): milled lactose (wherein notgreater than 85% of particles 12 mg have a MMD of 60-90 μm, and not lessthan 15% of particles have a MMD of less than 15 μm):

[0343] A peelable blister strip containing 60 blisters each filled witha formulation as just described may be prepared.

Example 46 Aerosol formulation containing6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0344] An aluminium canister may be filled with a formulation asfollows: 6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl- 250 μg1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β- carbothioicacid S-fluoromethyl ester (prepared according to Example 41 andmicronised to a MMD of 3 μm): 1,1,1,2-tetrafluoroethane: to 50 μl

[0345] (amounts per actuation)

[0346] in a total amount suitable for 120 actuations and the canistermay be fitted with a metering valve adapted to dispense 50 μl peractuation.

Example 47 Aerosol formulation containing6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester and a long acting β₂-adrenoreceptor agonist

[0347] An aluminium canister may be filled with a formulation asfollows: 6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl- 250 μg1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β- carbothioicacid S-fluoromethyl ester (prepared according to Example 41 andmicronised to a MMD of 3 μm): Long-acting β₂-adrenoreceptor agonist(micronised to a MMD 25 μg of 3 μm): 1,1,1,2-tetrafluoroethane: to 50 μl

[0348] (amounts per actuation)

[0349] in a total amount suitable for 120 actuations and the canistermay be fitted with a metering valve adapted to dispense 50 μl peractuation.

Example 49 Nasal formulation containing6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester

[0350] A formulation for intranasal delivery may be prepared as follows:6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl- 10 mg1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β- carbothioicacid S-fluoromethyl ester (prepared according to Example 41,micronised): Polysorbate 20 0.8 mg Sorbitan monolaurate 0.09 mg Sodiumdihydrogen phosphate dihydrate 94 mg Dibasic sodium phosphate anhydrous17.5 mg Sodium chloride 48 mg Demineralised water to 10 ml

[0351] The formulation may be fitted into a spraypump capable ofdelivering a plurality of metered doses (Valois).

[0352] Pharmacological Activity

[0353] In Vitro Pharmacological Activity

[0354] Pharmacological activity was assessed in a functional in vitroassay of glucocorticoid agonist activity which is generally predictiveof anti-inflammatory or anti-allergic activity in vivo.

[0355] The functional assay was based on that described by K. P. Ray etal., Biochem J. (1997), 328, 707-715. A549 cells stably transfected witha reporter gene containing the NF-κB responsive elements from the ELAMgene promoter coupled to sPAP (secreted alkaline phosphatase) weretreated with test compounds at appropriate doses for 1 hour at 37° C.The cells were then stimulated with tumour necrosis factor (TNF, 10ng/ml) for 16 hours, at which time the amount of alkaline phosphataseproduced is measured by a standard colourimetric assay. Dose responsecurves were constructed from which EC₅₀ values were estimated.

[0356] In this test the compounds of Examples 1 to 9, 11-13, 15-22 and24-42 showed an EC₅₀ value of <1 nM. In this test the compounds ofExamples 10, 14 and 23 showed an EC50 value of 1, 2 and 17 nMrespectively.

[0357] The glucocorticoid receptor (GR) can function in at least twodistinct mechanisms, by upregulating gene expression through the directbinding of GR to specific sequences in gene promoters, and bydownregulating gene expression that is being driven by othertranscription factors (such as NFκB or AP-1) through their directinteraction with GR.

[0358] In a variant of the above method, to monitor these functions, tworeporter plasmids have been generated and introduced separately intoA549 human lung epithelial cells by transfection. The first cell linecontains the firefly luciferase reporter gene under the control of asynthetic promoter that specifically responds to activation of thetranscription factor NFκB when stimulated with TNFα. The second cellline contains the renilla luciferase reporter gene under the control ofa synthetic promotor that comprises 3 copies of the consensusglucocorticoid response element, and which responds to directstimulation by glucocorticoids. Simultaneous measurement oftransactivation and transrepression was conducted by mixing the two celllines in a 1:1 ratio in 96 well plate (40,000 cells per well) andgrowing overnight at 37° C. Test compounds were dissolved in DMSO, andadded to the cells at a final DMSO concentration of 0.7%. Afterincubation for 1 h 0.5 ng/ml TNFα (R&D Systems) was added and after afurther 15 hours at 37° C., the levels of firefly and renilla luciferasewere measured using the Packard Firelite kit following themanufacturers' directions. Dose response curves were constructed fromwhich EC₅₀ values were determined. Transactivation (GR) Transrepression(NFκB) ED₅₀ (nM) ED₅₀ (nM) Example 1 0.06 0.20 Metabolite (X) >250 >1000Fluticasone propionate 0.07 0.16

[0359] Screen for progesterone receptor activity

[0360] The human breast cancer cell line T47D has been reported toupregulate an endogenous alkaline phosphatase in response to progestins(Di Lorenzo et al., Cancer Research (1991) 51, 4470-4475. T47D cellswere seeded into 96 well plates at a density of 1×10⁵ cells per well andgrown overnight at 37° C. Steroids were dissolved in DMSO, added to thecells (final DMSO concentration 0.7%), and incubated for 24 hours at 37°C. The cells were then washed with PBS and lysed with RIPA buffer (1%IGEPAL, 0.5% Na deoxycholate, 0.1% SDS in phosphate buffered saline).Alkaline phosphatase activity was measured spectrophotometrically (405nm) using p-nitrophenylphosphate (1.5 mg/ml) as a substrate dissolved in1 M diethanolamine, 0.28M NaCl, 0.5 mM MgCl₂. Dose response curves wereconstructed from which EC₅₀ values were estimated.

[0361] Examples 12 and 41 were tested for progesterone activity inaccordance with the above screen and the selectivity was determined bydividing the ED₅₀ at the progesterone receptor by the ED₅₀ at theglucocorticoid receptor.

[0362] The selectivity of Example 12 was determined to be 364 (comparefluticasone propionate: selectivity=23)

[0363] In separate experiment, the selectivity of Example 41 wasdetermined to be 1599 (compare fluticasone propionate: selectivity=60)

[0364] In Vivo Pharmacological Activity

[0365] Pharmacological activity in vivo was assessed in an ovalbuminsensitised Brown Norway rat eosinophilia model. This model is designedto mimic allergen induced lung eosinophilia, a major component of lunginflammation in asthma.

[0366] Example 1 produced dose dependant inhibition of lung eosinophiliain this model after dosing as an intra-tracheal (IT) suspension insaline 30 min prior to ovalbumin challenge. Significant inhibition isachieved after a single dose of 30μg of Example 1 and the response wassignificantly (p=0.016) greater than that seen with an equivalent doseof fluticasone propionate in the same study (69% inhibition with Example1 vs 41% inhibition with fluticasone propionate).

[0367] In a rat model of thymus involution 3 daily IT doses of 100 μg ofExample 1 induced significantly smaller reductions in thymus weight(p=0.004) than an equivalent dose of fluticasone propionate in the samestudy (67% reduction of thymus weight with Example 1 vs 78% reductionwith fluticasone propionate).

[0368] Taken together these results indicate a superior therapeuticindex for Example 1 compared to fluticasone propionate.

[0369] In vitro Metabolism in Rat and Human Hepatocytes

[0370] Incubation of Example 1 with rat or human hepatocytes shows thecompound to be metabolised in an identical manner to fluticasonepropionate with the 17-βcarboxylic acid (X) being the only significantmetabolite produced. Investigation of the rate of appearance of thismetabolite on incubation of Example 1 with human hepatocytes (37° C., 10μM drug concentration, hepatocytes from 3 subjects, 0.2 and 0.7 millioncells/mL) shows Example 1 to be metabolised ca. 5-fold more rapidly thanfluticasone propionate: Subject Cell density 17-β acid metaboliteproduction (pmol/h) number (million cells/mL) Example 1 Fluticasonepropionate 1 0.2 48.9 18.8 1 0.7 73.3 35.4 2 0.2 118 9.7 2 0.7 903 23.73 0.2 102 6.6 3 0.7 580 23.9

[0371] Median metabolite production 102-118 pmol/h for Example 1 and18.8-23.0 pmol/h for fluticasone propionate.

[0372] Pharmacokinetics After Intravenous (IV) and Oral Dosing in Rats

[0373] Examples 1 and 41 were dosed orally (0.1 mg/kg) and IV (0.1mg/kg) to male Wistar Han rats and pharmacokinetic parametersdetermined. Example 1 showed negligible oral bioavailability (0.9%) andplasma clearance of 47.3 mL/min/kg, approaching liver blood flow (plasmaclearance of fluticasone propionate =45.2 mL/min/kg).

[0374] Examples 4,19, 24, 25, 28 and 41 were dosed IV (0.1 mg/kg) tomale Wistar Han rats and plasma clearance values of 49, 48, 47, 46, 51and 42 mL/min/kg respectively were determined.

[0375] Pharmacokinetics After Intra-Tracheal Dry Powder Dosing in thePig.

[0376] Anaesthetised pigs (2) were dosed intra-tracheally with ahomogenous mixture of Example 1 (1 mg) and fluticasone propionate (1 mg)as a dry powder blend in lactose (10% w/w). Serial blood samples weretaken for up to 8 h following dosing. Plasma levels of Example 1 andfluticasone propionate were determined following extraction and analysisusing LC-MS/MS methodology, the lower limits of quantitation of themethods were 10 and 20pg/mL for Example 1 and fluticasone propionaterespectively. Using these methods Example 1 was quantifiable up to 2hours after dosing and fluticasone propionate was quantifiable up to 8hours after dosing. Maximum plasma concentrations were observed for bothcompounds within 15 min after dosing. Plasma half-life data obtainedfrom IV dosing (0.1 mg/kg) was used to calculate AUC (0-inf) values forExample 1. This compensates for the plasma profile of Example 1 onlybeing defined up to 2 hours after an IT dose and removes any bias due tolimited data between Example 1 and fluticasone propionate.

[0377] C_(max) and AUC (0-inf) values show markedly reduced systemicexposure to Example 1 compared to fluticasone propionate: Cmax (pg/mL)AUC (0-inf) (hr.pg/mL) Pig 1 Pig 2 Pig 1 Pig 2 Example 1 117  81 254 221Fluticasone propionate 277 218 455 495

[0378] The pharmacokinetic parameters for both Example 1 and fluticasonepropionate were the same in the anaesthetised pig following intravenousadministration of a mixture of the two compounds at 0.1 mg/kg. Theclearance of these two glucocortocoids is similar is this experimentalpig model.

[0379] Throughout the specification and the claims which follow, unlessthe context requires otherwise, the word ‘comprise’, and variations suchas ‘comprises’ and ‘comprising’, will be understood to imply theinclusion of a stated integer or step or group of integers but not tothe exclusion of any other integer or step or group of integers orsteps.

[0380] The patents and patent applications described in this applicationare herein incorporated by reference.

1. A pharmaceutical formulation for administration by inhalationcomprising a compound of formula (I),

wherein R₁ represents C₁₋₆ alkyl or C₁₋₆ haloalkyl; R₂ represents—C(═O)-aryl or —C(═O)-heteroaryl; R₃ represents hydrogen, methyl (whichmay be in either the α or β configuration) or methylene; R₄ and R₅ arethe same or different and each represents hydrogen or halogen; and

represents a single or a double bond; or a salt or solvate thereof,together with a long-acting β₂-adrenoreceptor agonist which formulationhas a therapeutically useful effect in the treatment of COPD over aperiod of 24 hours or more.
 2. A pharmaceutical formulation according toclaim 1 wherein the compound of formula (I) or a solvate thereof and thelong-acting β₂-adrenoreceptor agonist are both present in particulateform.
 3. A pharmaceutical formulation according to claim 2 furthercomprising a particulate carrier.
 4. A pharmaceutical formulationaccording to claim 3 wherein the carrier is lactose.
 5. A pharmaceuticalformulation according to claim 1 further comprising a liquifiedpropellant gas.
 6. A pharmaceutical formulation according to claim 2further comprising a liquified propellant gas.
 7. A method of treatmentof COPD once-per-day which comprises administration of a pharmaceuticalformulation according to claim
 1. 8. An inhaler containing a pluralityof doses of a pharmaceutical formulation comprising a compound offormula (I)

wherein R₁ represents C₁₋₆ alkyl or C₁₋₆ haloalkyl; R₂ represents—C(═O)-aryl or —C(═O)-heteroaryl; R₃ represents hydrogen, methyl (whichmay be in either the α or β configuration) or methylene; R₄ and R₅ arethe same or different and each represents hydrogen or halogen; and

represents a single or a double bond; or a salt or solvate thereof,together with a long-acting β₂-adrenoreceptor agonist which formulationhas a therapeutically useful effect in the treatment of COPD over aperiod of 24 hours or more, and which doses are suitable foronce-per-day administration of the formulation by inhalation.
 9. Aninhaler according to claim 8 wherein the compound of formula (I) or asolvate thereof and the long-acting β₂-adrenoreceptor agonist are bothpresent in particulate form.
 10. An inhaler according to claim 9 whereinthe formulation further comprises a particulate carrier.
 11. An inhaleraccording to claim 10 wherein the carrier is lactose.
 12. An inhaleraccording to claim 8 wherein the formulation further comprises aliquefied propellant gas.
 13. An inhaler according to claim 9 whereinthe formulation further comprises a liquefied propellant gas.
 14. Aninhaler containing a plurality of doses of a pharmaceutical formulationcomprising a particulate compound of formula (I)

wherein R₁ represents C₁₋₆ alkyl or C₁₋₆ haloalkyl; R₂ represents—C(═O)-aryl or —C(═O)-heteroaryl; R₃ represents hydrogen, methyl (whichmay be in either the α or β configuration) or methylene; R₄ and R₅ arethe same or different and each represents hydrogen or halogen; and

represents a single or a double bond; or a salt or solvate thereof,together with a long-acting β₂-adrenoreceptor, a particulate long-actingβ₂-adrenoreceptor agonist and a carrier each drug being present in anamount adequate to provide a therapeutically useful effect in thetreatment of COPD over a period of 24 hours or more followingonce-per-day dosing by inhalation.
 15. A formulation according to claim1 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 16. Aformulation according to claim 2 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 17. Aformulation according to claim 3 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 18. Aformulation according to claim 4 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 19. Aformulation according to claim 5 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 20. Aformulation according to claim 6 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 21. A methodaccording to claim 7 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 22. An inhaleraccording to claim 8 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 23. An inhaleraccording to claim 9 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 24. An inhaleraccording to claim 10 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 25. An inhaleraccording to claim 11 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 26. An inhaleraccording to claim 12 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 27. An inhaleraccording to claim 13 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.
 28. An inhaleraccording to claim 14 wherein the compound of formula (I) is6α,9α-Difluoro-11β-hydroxy-16α-methyl-17β-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a salt or solvate thereof.